PROPERTIES OF GROWTH SUBSTANCES 41 



filter paper contained the growth substance, which Nielsen named 

 rhizopin. (5) Rhizopin is soluble in water, alcohol, 90 per cent 

 acetone, and ether. It can be extracted from aqueous solution 

 with ether but not with xylene or benzene. It is very sensitive 

 to peroxide. 



The ether should be freed from peroxides before use. Its 

 purification may be accomplished by the Garbarini method, as 

 follows: 2 liters of ether are shaken up with 1 g. calcium hydroxide 

 and 3 g. ferrous sulphate in 20 cc. water. The ether is then 

 distilled. Rliizopin soon becomes ineffective if dissolved in 

 ether that has not been purified in some such way as this. 



The Identity of Rhizopin and S-Indole Acetic Acid. — Growth 

 substance prepared from Rhizopus and Aspergillus was shown 

 (Kogl and Kostermans, 1934, Mitt. XIII) to have a molecular 

 weight close to that of 3-indole acetic acid, and Thimann (19356) 

 showed that the active substance ''rhizopin" is almost certainly 

 identical with 3-indole acetic acid; in the same paper, Thimann 

 outlined the steps for purification of this substance extracted from 

 Rhizopus. 



Aspergillus, a Source of Growth Substance. — Boysen Jensen 

 (19316) showed thsLt Aspergillus nigeriorms growth substance when 

 cultured on a fluid substratum, but only when peptone or haemo- 

 globin is present as a source of nitrogen. The method of 

 preparation was as follows: 



(1) Large covered culture dishes of tin plate, 37 by 58 cm., 

 were used for growing the fungus. The dishes were coated on the 

 inside with a solution of paraffin in petroleum ether. (2) Each 

 culture vessel contained about 3 liters glucose-peptone solution 

 (1.5 cm. in depth). Each liter of solution contained 20 g. impure 

 glucose, 5 g. peptone, 5 g. citric acid, and 0.25 g. monobasic 

 potassium phosphate added to distilled water. (3) A layer 

 (2 to 5 mm. in depth) of sterilized cork particles was placed on the 

 culture fluid to form a substratum for the fungus. (4) The cul- 

 ture medium was inoculated with Aspergillus spores which had 

 been soaked in sterilized water. (5) The temperature was 

 maintained for 3 days at 33 to 34°C. If the culture showed no 

 infection at the end of this time, the temperature was raised to 

 36 to 37°C. for the next 7 days. The production of growth sub- 

 stance apparently takes place chiefly after the cessation of growth. 

 When the reaction of the culture fluid becomes alkahne, the 



